Considerable attention has been directed in recent years to the design of antisense nucleic acid oligomers for use in studying, treating and diagnosing conditions attributable to endogenous or foreign nucleic acid sequences in living organisms. For example, it is now well known that a nucleic acid oligomer having suitable antisense complementarily to a target mRNA can hybridize to the target mRNA and, in some cases, disrupt translation of the mRNA. The antisense approach presents great promise for the eventual therapeutic treatment of disease conditions caused by foreign (e.g., viral) genetic material, or by misfunctioning or altered endogenous genetic material (e.g., cancer and genetic disease conditions).
However, despite the great promise of the antisense approach, a number of challenges still remain. First, antisense compounds are generally subject to degradation in the cellular milieu due to endogenous endo- and exonucleases. While a number of modified antisense structures have been described having improved resistance to nuclease degradation, further improvements are desirable in order to increase the potency and half-life of the compounds. Second, it is generally required that an antisense compound have a high specificity toward the intended target nucleic acid so as to avoid disruption of activity of unintended native sequences. Although a number of researchers have described approaches designed to increase the binding affinity of an antisense compound to a target sequence, very few results have been reported with respect to structural refinements which avoid disruption of the activity of unintended genetic sequences while still retaining maximum efficacy against the target sequence.
One approach toward disrupting the expression of undesired target mRNAs involves forming a duplex hybrid between the target mRNA and an antisense strand, followed by cleavage of the target mRNA by an endogenous RNaseH. See Dash, P., et al, Proc. Natl. Acad. Sci. U.S.A. 84:7896-7990 (1987). However, because the mode of action of RNaseH is fairly specific, this approach is subject to a number of constraints. First, RNaseH enzymes act in nature to cleave the oligoribonucleic acid strand of an oligodeoxyribonucleotide-oligoribonucleotide duplex, but do not cleave DNA-DNA or RNA-RNA duplexes. This has been attributed, at least in part, to the polar nature of DNA-RNA hybrids which, in contrast to DNA-DNA and RNA-RNA hybrids, have 2'-OH groups on one (but only one) strand. Crouch, R. J. & Dirksen, M.-L., "Ribonucleases H," in Nucleases (Linn & Roberts, eds.), Cold spring Harbor Laboratory (1982), at 212. As a result, one putative requirement of the antisense RNaseH cleavage approach is that at least some of the nucleosides of the antisense nucleic acid strand must have characteristics in common with deoxyribonucleotides (as opposed to ribonucleotides), particularly, the absence of a polar group on the 2'-position of the antisense nucleoside sugars. Perhaps related to this is the additional requirement that at least some of the sugar groups in the antisense compound must be in a 2'-endo (.beta.) conformation as found in deoxyribonucleosides, as opposed to the 3'-endo (.alpha.) conformation found in ribonucleosides. Cook P. D., PCT Publication No. WO 93/13121 (1993), at 18-19.
It has further been reported that various 2'-position substituents (e.g., 2'-O-alkyl and 2'-fluoro) will render the substituted portion of an antisense strand non-activating to RNaseH, even though binding affinity toward the target nucleic acid is increased. Inoue, H., et al., FEBS Letters 215(2):327-330 (1987); Monia, B. P., et al., J. Biol. Chem. 268(19):14514-14522 (1993). Likewise, the Monia, et al. report indicates that a minimum of five consecutive 2'-deoxy residues is required in order to achieve efficient activation of mammalian (HeLa) RNAseH, and that this 2'-deoxy segment (if accompanied by 2'-substituted residues in the same antisense compound) must be centered in the oligomer sequence in order to achieve efficient RNaseH activation in vitro or expression inhibition in cells.
Another reported requirement of the antisense RNaseH cleavage approach is that, in order to achieve RNaseH activation, at least one portion of the internucleoside "backbone" of the antisense compound must include charged (anionic) phosphorus-containing linkage groups. Cook, P. D., PCT Publication No. WO 93/13121 (1993), at 18. In studies of chimeric antisense compounds including both methylphosphonate (uncharged) and phosphodiester or phosphorothioate (charged) linkages, Agrawal, et al. reported that the minimum number of consecutive charged backbone linkages required for efficient activation of mammalian RNaseH in vitro is five. Phosphodiester linkages positioned in either the terminal or center portion of the oligomers were reportedly more efficient than phosphorothioate linkages in activating RNaseH, whereas oligomers containing only methylphosphonate, phosphoro-N-morpholidate or phosphoro-N-butylamidate linkages were inactive. Agrawal, S., et al., Proc. Natl. Acad. Sci. U.S.A. 87:1401-1405 (1990).
While phosphodiester linkages, being charged, are suitable to allow activation of RNaseH, they suffer from the disadvantage of being subject to degradation by naturally-occurring endo- and/or exonucleases. A variety of alternative linkage groups, some of which are nuclease-resistant, have been developed or proposed for use with antisense compounds. Among these are charged linkage groups such as phosphorothioate, phosphorodithioate, phosphoroselenate and phosphorodiselenate linkers. In general, deoxyribonucleoside antisense oligomers containing these non-natural linkage groups tend to have lower binding affinity toward complementary RNA target strands than the corresponding phosphodiester-linked antisense oligomers, although higher affinity may be achieved where the antisense strand comprises ribonucleosides or 2'-substituted ribonucleosides (rather than deoxyribonucleosides). See Metelev, V. & Agrawal, S., PCT Publication No. WO 94/02498 (1994), at 9. Among the uncharged phosphorus-containing linkage groups that have been reported are the alkylphosphonate (e.g., methylphosphonate), aryl phosphonate, alkyl and aryl phosphoramidate, alkyl and aryl phosphotriester, hydrogen phosphonate, boranophosphate, alkyl and aryl phosphonothioate, phosphoromorpholidate, and phosphoropiperazidate linkers. See Cook, P. D., PCT Publication No. WO 93/13121 (1993), at 7; Pederson, T., et al., U.S. Pat. Nos. 5,149,797 and 5,220,007; Padmapriya, A. & Agrawal S., PCT Publication No. WO 94/02499 (1994). Non-phosphorus-based linkage groups have also been reported, including peptide, morpholino, ethylene glycol, amide, and other linkers. See Reynolds, M. A., et al., PCT Publication No. WO 92/02532 (1992); Cook, P. D., PCT Publication No. WO 93/13121 (1993), at 7. As with the charged phosphorus-containing linkers noted above, many of these other non-natural linkage groups may exhibit lower binding affinity (compared to phosphodiester linkages) toward complementary RNA target strands, at least in the case of linked 2'-unsubstituted antisense nucleotides, and particularly in the presence of salt ions.
Various workers have attempted to identify combinations of linkage groups and/or structural modifications for antisense oligomers that might lead to improved RNaseH activation, binding affinity, nuclease resistance and/or target specificity. Thus, Cohen, et al. have reported improved half-life for antisense and non-antisense oligodeoxyribonucleotides containing at least one phosphorothioate linkage located, for example, at either terminus of the compound, or throughout the compound. Oligomers containing all phosphorothioate linkages were shown to have anti-viral (anti-HIV) activity, whereas phosphodiester- and methylphosphonate-linked compounds were reportedly inactive. Cohen, J. S., et al., U.S. Pat. No. 5,264,423. Walder et al. have proposed the use of a 3'-terminal non-phosphodiester linkage, optionally combined with a 5'-terminal non-phosphodiester linkage or a 5'-terminal "cap" group, to avoid 3'-initiated (and optionally 5'-initiated) exonuclease degradation of oligodeoxyribonucleotides. RNaseH cleavage activation reportedly required retention of at least four, and preferably at least seven, contiguous phosphodiester linkages in the antisense oligomer. The preferred compounds contained at least 10, and preferably at least 15, nucleotides, the majority of which were phosphodiester-linked. Walder, J. A., et al., PCT Publication No. WO 89/05358 (1989). Padmapraya & Agrawal have reported that the incorporation of nonionic alkyl or aryl phosphonothioate linkages, preferably at one or both termini of the oligomer, resulted in improved nuclease resistance, albeit with a reduction in Tm of 1-2.degree. C./phosphonothioate linkage. PCT Publication No. WO 94/02499 (1994).
Pederson, et al. have reported the use of "mixed phosphate backbone" oligomers containing both a phosphodiester- or phosphorothioate-linked segment for RNaseH activation, and one or more non-RNaseH-activating, uncharged linkage group segments. It was found that a segment of five or six consecutive phosphodiester linkages was efficient, in a 15-mer compound, to effect RNaseH cleavage of a target RNA strand, whereas similar compounds with fewer phosphodiester linkages, or with up to six consecutive phosphorothioate linkages in place of the phosphodiester linkages, had low activity. Pederson, T., et al., U.S. Pat. Nos. 5,149,797 and 5,220,007.
Giles & Tidd have reported that the target specificity of an antisense oligomer can be improved by the use of a chimeric structure comprising terminal methylphosphonodiester sections separated by a central RNaseH-activating phosphodiester region having a high A+T to G+C ratio. The observed reductions in non-specific cleavage were attributed to the lower Tm caused by the methylphosphonate segments, the reduced hybridization strength of the small, A/T-rich phosphodiester region, and the reduced prospects for partially-complementary hybridization at the shortened RNaseH activation site. Giles, R. V. & Tidd, D. M., Nucl. Acids Res. 20(4):763-770 (1992).
Ohtsuka, et al. have described the use of partially 2'-substituted (e.g., 2'-lower alkoxy substituted) oligomers for site-specific RNaseH cleavage of RNA targets with or without secondary structure. RNaseH cleavage was reportedly localized to a site (or sites) on the target corresponding to the non-substituted (i.e., deoxyribonucleotide) portion of the antisense compound. Single-site cleavage was reportedly optimized by use of a tetradeoxyribonucleotide segment located centrally in the compound between two 2'-substituted terminal segments. Inoue, H., et al., FEB Letters 215(2):327-330 (1987); Shibahara, S., et al., Nucl. Acids Res. 15(11):4403-4415 (1987); Ohtsuka, E., et al., U.S. Pat. No. 5,013,830. The use of partially 2'-substituted oligomers additionally containing one or more non-phosphodiester linkages has also been reported. See Shibahara, S., et al., European Patent Application Publication No. 0 339 842 A2 (1989) (reporting 3'-5' or 2'-5' linked oligomers having phosphorothioate or other linkages); Cook, P. D., PCT Publication No. WO 93/13121 (1993) (reporting increased binding affinity attributable to 2'-substitutions, and nuclease resistance attributable to, e.g., phosphorothioate and phosphorodithioate linkages); Monia, B. P., et al., J. Biol. Chem. 268(19):14514-14522 (1993) (reporting effects of 2'-substitutions in phosphorothioate-linked oligomers); Metelev, V. & Agrawal, S., PCT Publication No. WO 94/02498 (1994) (reporting use of 2'-substitutions in phosphorothioate- or phosphorodithioate-linked oligomers); McGee, D. P., et al., PCT Publication No. WO 94/02501 (1994) (describing preparation of various 2'-substituted nucleosides and phosphoramidites).